Method for preparing particles from an emulsion in supercritical or liquid co2

ABSTRACT

The present invention relates to a method for preparing particles, notably particles encapsulating an active substance. It also relates to particles obtainable by this process, dispersion thereof, and their use as a vehicle for pharmaceutical, cosmetic, diagnostic, veterinary, phytosanitary active substances or processed foodstuff.

The present invention relates to a method for preparing particles,notably particles encapsulating an active substance.

It also relates to particles obtainable by this process, dispersionthereof and their use as a charge or as a vehicle for pharmaceuticals,cosmetics, diagnostic compositions or processed foodstuff.

The encapsulation of substances into micro or nanoparticles has receivedan increased interest over the last years for various applications indiverse fields like medicine, chemistry, cosmetics, or nutrition. Forinstance, in the pharmaceutical field, the incorporation of thepharmacologically active molecules into microspheres allows to controltheir spatial and temporal delivery into the human body and permits toprotect them against degradation.

The encapsulation processes used in pharmaceutical industry must takeinto account several requirements such as the stability of the activemolecules, the yield and the efficiency of the drug encapsulation, thereproducibility of the microspheres quality and of the drug releaseprofile, and the residual level of organic solvent in themicroparticles, which should be lower than the limit value imposed bythe Pharmacopoeia.

However, the common techniques used by the pharmaceutical industry toencapsulate active molecules, like emulsification-solvent removal,polymer phase-separation, spray drying and milling methods are notalways suitable to formulate particles within these requirements(Arshady et al., 1991; Benoit et al., 1996). They often use harshformulation conditions, which can induce physical or chemical changes inthe active substances, especially if it is a protein, and lead toamounts of residual solvent that are difficult to decrease under theupper authorized values. In fact, the volatile organic chemicals (VOC),usually used in these processes, are subject to very strictinternational regulations (ICH Harmonized Tripartite Guideline forResidual Solvents, 1997), due to their toxicity for humans and for theenvironment.

Therefore, in the need to find new encapsulation methods, which offer agood encapsulation yield, do not damage the structure of the activesubstances and do not use any organic solvents, some researches areoriented to alternative methods, such as working with compressed carbondioxide instead of the organic solvents. As a matter of fact, the carbondioxide is non-toxic, non-flammable, abundant, recyclable,environmentally friendly and it has a tunable solvent power when it isclose to its critical point (Tc=31.1° C. and Pc=73.8 bar). Due to itsproperties, it is neither regulated as a VOC, and nor restricted in foodor pharmaceutical applications. All these advantages made compressed CO₂an attractive solvent that offers a “green” alternative to thetraditional organic solvents already used in a lot of processes such asextraction (Saldana et al., 2002), separation (Mendes et al., 2003;Ozcan et al., 2004), fractionation, cleaning (Campbell et al., 2001),reaction medium (Jacobson et al., 1999; Kane et al., 2000) or phase foremulsions-microemulsions (Psathas et al., 2002; Eastoe et al., 1997;Hoefling et al., 1991; Lee et al., 1999).

Consequently, the researches on the properties and possible applicationsof the compressed CO₂ have rapidly progressed over the last ten years.In this respect, much research has been done on water-carbon dioxide(W—C) binary systems (Eastoe et al., 1997, Bartscherer et al., 1995;Harrison et al., 1994; da Rocha et al., 2003; Johnston et al., 2001;Psathas et al., 2000; Ryoo et al., 2003; Zielinski et al., 2004), asthey offer the advantage of acting as “universal” solvents, due to theirbinary composition allowing the dissolution of either polar or lowmolecular weight apolar substances.

Among the methods for preparing particles using supercritical orpressurised fluids, mention may be made of (i) technique using thesupercritical fluids (hereafter called SF) as a solvent to solubilizeactive and/or carrier molecules, such as the Rapid Expansion ofSupercritical Solution (RESS) technique, (ii) techniques using the SF asan antisolvent, where it is brought into contact with an organicsolution to induce precipitation of the active molecules and/or thecarrier, such the Gas Anti-Solvent (GAS) and Supercritical Anti-Solvent(SAS) related techniques, and (iii) techniques using the SF as a sprayenhancer such as the techniques named Particles from Gas SaturatedSolutions (PGSS), Polymer Liquefaction Using Supercritical Solvation(PLUSS), Supercritical-Assisted Atomization (SAA), Depressurization ofan Expanded Liquid Organic solution (DELOS), CO₂-Assisted Nebulizationand Bubble Drying (CAN-BD).

Thus, RESS process can be used when the substance of interest is highlysoluble in the supercritical phase. The substance is dissolved in thesupercritical phase and the formed solution is expanded rapidly bydepressurizing the system through a heated nozzle, so that the substanceprecipitates as very small particles.

The GAS and SAS processes can be used for crystallization of substanceswhich are not or only slightly soluble in the supercritical fluid. Forthe GAS processes, the substance of interest is dissolved in the organicsolvent. When the supercritical fluid, which has a low solvent capacitywith respect to the solute(s) but is completely miscible with theorganic solvent, is added to a batch of the organic solution, thesolute(s) precipitates in particles because the CO₂-expanded solvent hasa lower solvent strength than the pure solvent.

For the SAS processes, the organic solution is sprayed through a nozzle,producing small solvent droplets, into a vessel filled of CO₂. Then, theCO₂ expands the solvent of the droplets, leading to formation ofparticles. Several adaptations of the SAS processes have been performedsuch as the ASES, PCA and the Solution Enhanced Dispersion bysupercritical fluid (SEDS) process.

Micronization of proteins and polymer microparticles has been preparedaccording to these methods.

The principle of the processes using CO₂ as a spray enhancer consists indissolving supercritical CO₂ (hereafter called SCCO₂) in meltedsubstance(s) or in a solution/suspension of substances anddepressurising this mixture through a nozzle, causing the formation ofsolid particles or liquid droplets. These processes allow particles toform from melt polymers that are not soluble in SCCO₂, but which absorba large amount of CO₂ (1-30 wt %) (Tomasko D. L. et al., 2003).

However, these methods of processing materials using supercriticalfluids hardly make it possible to control the morphology and the size ofthe obtained particles. Finally, a large number of these methods furtheruses organic co-solvents that may raise environmental and safetyproblems.

The aim of the present invention is to provide a method for preparingparticles having a controlled size and morphology without the use oftoxic organic solvent.

Thus, in one aspect, the invention is directed to a method for preparingparticles comprising the steps of:

i) preparing an emulsion containing

-   -   as a continuous phase, supercritical or liquid CO₂, and    -   as a discontinuous phase, a solvent containing a substance        selected from the group consisting of a polymer, a cationic        divalent ion or a mixture thereof;    -   said substance being soluble in said solvent and insoluble in        said continuous phase;

ii) solidifying said discontinuous phase, thereby forming particles.

The inventors have now demonstrated that it is possible to obtainspherical and uniform size particles by emulsifying a solution of asubstance selected from the group consisting of a polymer, a cationicdivalent ion or a mixture thereof in liquid or supercritical CO₂, beforethe solidification phenomenon starts. In this process, emulsion dropletsact as templates for the obtaining of the particles, by guiding thesolidification process and limiting the solidification extent.

Advantageously, the method of the invention allows to reduce theaggregation of particles.

In accordance with another advantage, particles can be produced by thismethod with high yield, and without use of VOC or high temperatureprocesses.

This method is particularly advantageous to prepare biocompatible andbiodegradable particles containing an active substance, which may beonly slightly recognised by the complement protein system and by themacrophages of the Mononuclear Phagocyte System (MPS).

Particles

As used herein, the term “particles” refers to an aggregated physicalunit of solid material.

The particles according to the invention may be micro- or nanoparticles.

Microparticles are understood as particles having a median diameter d₅₀ranging from 500 μm to 1 μm and more preferably from 100 μm to 1 μm, andmost preferably from 10 μm to 1 μm.

Nanoparticles are understood as particles having a median diameter d₅₀inferior to 1 μm and notably ranging from 0.1 μm and 0.01 μm.

As used herein, the term “median diameter d₅₀” refers to the particlediameter so that 50% of the volume or of the number of the particlespopulation have a smaller diameter.

The d₅₀ of the particles prepared according to the invention from anemulsion containing divalent cationic ions is generally expressed bynumber of particles.

The d₅₀ of the other particles is generally expressed by volume.

The particles may also be defined by their mean diameter. As usedherein, the term “mean diameter” refers to the sum of the sizemeasurements of all measurable particles measured divided by the totalnumber of particles measured.

The mean and the median diameter d₅₀ according to the invention aredetermined by virtue of a particle size measurement performed on thesuspensions according to the method based either on “Light Diffusion” oron the “Variation of impedance between two electrodes”.

More specifically, the microparticles or nanoparticles may bemicrospheres or microcapsules, nanospheres or nanocapsules respectively,containing an active substance.

Emulsion

As used herein, “microspheres” or “nanospheres” are matrix systems inwhich the active substance is homogeneously dispersed.

“Microcapsules” or “nanocapsules” are composed of a nucleus of activematerial coated with a layer of polymer.

As used herein, the term “emulsion” refers to a heterogeneous system ofone immiscible liquid (discontinuous phase) dispersed in another fluidor liquid in the form of droplets. The size of the droplets of theemulsion may range from 10 nm to several μm, for example to 500 μm.Thus, the term “emulsion” in the context of the present inventionencompasses notably miniemulsions, nanoemulsions, macroemulsions andmicroemulsions.

In the context of the present invention, the emulsion may be notably aliquid-liquid emulsion, or a liquid-supercritical fluid emulsion, thatmeans that the CO₂ may be in supercritical conditions or in liquidstate.

CO₂ is said to be in the supercritical conditions (SC CO₂) if thetemperature is greater than 31° C. and its pressure greater than73.8×10⁵ Pa. SC CO₂ presents some properties of both liquid and gasphases. Thus, above the critical point (Tc, Pc), i.e. between Tc-1.2Tcand Pc-2Pc, the fluid properties, like density (ρ, kg/m³), varyaccording to a continuum from liquid-like to gas-like. At constanttemperature, an increase in pressure results in an increase in density,while for a constant pressure, an increase in temperature leads to adecrease in the density, which remains however close to a liquiddensity. Consequently, the density and thus the solvent power of thesupercritical fluid are tuneable in a small range of pressure andtemperature. The viscosity (η, Pa·s) varies like the density, but it isless dependent on temperature at constant pressure and it is 10-100times lower than the liquid density.

The interfacial tension (γ mN/m) between CO₂, notably SC CO₂, and thediscontinuous phase is relatively low, compared with classicalinterfaces (oil/water) and can be tuned by the fluid density, that is bytuning pressure and temperature conditions. More the density is high andmore the fluid develops interactions with the second substance, leadingto a decrease of the interfacial tension γ.

Thus, the emulsion may be prepared by contacting a solvent containing asubstance selected from the group consisting of a polymer, a cationicdivalent ion or a mixture thereof, soluble in said solvent, with CO₂,under temperature and/or pressure conditions allowing to decrease theinterfacial tension and thus to obtain an emulsion of the liquidsolution in the CO₂ phase.

The emulsion may be prepared by any conventional methods which mayinclude notably shearing, high pressure homogenization, static mixing,sonication, phase inversion induced by temperature or/and pressure.

Preferably, the solvent containing said substance is brought intocontact with CO₂ under stirring. The stirring may be effected with amoving blade, such as an anchor or a propeller moving blade. Thestirring rate may vary in a large range and is generally comprisedbetween 200 and 2000 rpm, notably between 500 and 1500 rpm.

Continuous Phase of the Emulsion

Preferably, the emulsion prepared at step i) contains as a continuousphase, supercritical CO₂.

The pressure of supercritical CO₂ may notably range from 80 to 350.10⁵Pa, preferably from 200 to 250.10⁵ Pa.

The temperature of the supercritical CO₂ may range from 32 to 50° C.,preferably from 35° to 45° C.

Advantageously, this temperature does not lead to a denaturation ofbiopolymers or derivatives thereof such as proteins forming theparticles of the invention.

Discontinuous Phase of the Emulsion

Substance Contained in the Discontinuous Phase

The substance contained in the discontinuous phase is soluble in thesolvent forming discontinuous phase and insoluble in CO₂ in theconditions of temperature and pressure of the process according to theinvention.

As used herein, the expression “the substance is soluble in the solventand insoluble in CO₂ phase” means a substance which is much more solublein the solvent than in the CO₂ phase. This can be estimated by thepartitioning coefficient (Kp), i.e. the ratio between the concentrationof the substance in the solvent and the concentration of the substanceinto CO₂. Typically, the substance exhibits a Kp higher than said 0.8.Hence, the term “insoluble” is not to be interpreted strictly and mayrefer to substances which are slightly soluble in the CO₂ phase.

The polymer may be selected among biopolymers or derivatives thereof orsynthetic polymers.

As used herein, the term “biopolymer” is understood to mean a moleculefound in nature, comprising more than 30 monomer units, typicallycomprising up to hundred of individual monomer units. Monomer units maybe notably sugars, amino acids and nucleotides.

In the context of the present invention, the term “biopolymer” alsoincludes the bio-oligomers which comprise 30 or less monomer units.

As examples of biopolymers, mention may be made of peptides, proteins(globular or fibrous) such as collagen (amino acid monomers),polysaccharides such as cellulose (sugar monomers), nucleic acid such asRNA and DNA (nucleotide monomers). Other examples of biopolymers arelignin and natural rubber.

Biopolymers may contain many different monomers such as amino acids, orconsist of one or two monomers repeated many times, for example,hyaluronic acid.

As used herein, the term “derivative of a biopolymer” or “modifiedpolymer” refers to biopolymers which have undergone a change on theirbackbone, such as, for example, the introduction of reactive functionalgroups (such as carboxyl groups or sulphuric ester groups) or thegrafting of chemical entities (molecules, fluorescent compounds,aliphatic links, PEG chains, and the like), or depolymerization byphysical, chemical or enzymatic methods.

Polysaccharides which are particularly suitable in the invention are orderive from D-glucose, D-galactose, D-mannose, D-fructose or fucose.

Examples of polysaccharides or derivatives thereof comprising one ormore D-glucose monomer units are notably:

-   -   cellulose or derivatives thereof such as carboxymethylcellulose,        ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose,        hydroxypropyl-cellulose, methylhydroxyethylcellulose or        methylhydroxypropylcellulose,    -   starch or derivatives thereof such as carboxymethylstarches,    -   dextran,    -   cyclodextrin.

Examples of polysaccharides or derivatives thereof comprising one ormore D-fructose monomer units are notably galactosan, mannan, fructosan.

An example of polysaccharides or derivatives thereof comprising one ormore fucose as monomer units is fucan.

The majority of these polysaccharides comprise the elements carbon,oxygen and hydrogen.

The polysaccharides in accordance with the invention can also comprisesulfur and/or nitrogen. They can thus derive from glycoprotein or fromglycolipid. Likewise, hyaluronic acid (composed of N-acetylglucosamineand glucuronic acid units), poly(sialic acid), also known as colominicacid or poly(N-acetyl-neuraminic acid), chitosan, chitin, heparin ororosomucoid comprise nitrogen, while agar, a polysaccharide extractedfrom marine algae, comprises sulfur in the form of hydrogen sulfate(>CH—O—SO₃H). Chondroitin sulfuric acid and heparin comprise both sulfurand nitrogen.

Other examples of polysaccharides or their derivatives are notably:

-   -   alginates extracted from brown algae,    -   carragheenans of lambda, iota of kappa type extracted from red        algae,    -   pectins extracted from lemons, apples or beetroot,    -   pectates which result from the demethylation of pectins,    -   guars or modified guars, such as carboxymethylguars,    -   xanthans.

Mention may be made, as illustration of the polysaccharides which aremore particularly suitable in the invention, of polydextroses, such asdextran, chitosan, pullulan, starch, amylose, amylopectin, cyclodextrin,hyaluronic acid, heparin, cellulose, pectin, alginate, curdlan, fucan,succinoglycan, chitin, xylan, xanthan, arabinan, carragheenan,poly(glucuronic acid), poly(N-acetyl-neuraminic acid), poly(mannuronicacid) and their derivatives (such as, for example, dextran sulfate,amylose esters, cellulose acetate, pentosan sulfate, and the like).

Examples of proteins or derivatives thereof include notably albumins,globulins, gelatin, casein, tubulin, actin, collagen, keratin, plantproteins (gliadin, glutenins, legumin, vicilin, convicilin, albumins).

Biopolymers are advantageously biodegradable and may be useful fortherapeutic uses.

As used herein, the term “synthetic polymer” refers to a large moleculetypically comprising up to thousand individual monomer units which maybe identical or different. Thus, the term “synthetic polymer” includeshomopolymers or copolymers.

Preferably, the synthetic polymer is biodegradable, which, according tothe invention, means a polymer in which the degradation may result fromthe action of natural chemical reactions like hydrolysis or with thehelp of procaryote or eucaryote cells.

According to another preferred embodiment, the polymer is biocompatible.

As used therein, “biocompatible polymer” refers to those polymers whichare, within the scope of sound medical judgement, suitable for contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio.

Examples of synthetic polymers include notably:

-   -   acrylic polymers or derivatives thereof such as polyacrylic or        polymethacrylic acid, polyesters of acrylic or methacrylic acid        (such as Poly(Methyl MethAcrylate) (PMMA)),    -   polyacrylamide,    -   polyalkylcyanoacrylates,    -   vinyl polymers and derivatives thereof such as polymers of vinyl        esters (for example, poly(vinyl acetate) or copolymers of        ethylene and of vinyl acetate), polyvinyl alcohols,    -   polyolefins such as polyethylene, polypropylene,    -   polyamides,    -   polyamides esters,    -   polyalkylenes esters,    -   poly(alpha-hydroxy acid)s,    -   poly(beta-hydroxy acid)s,    -   polylactic acid and its copolymers, such as homopolymers and        copolymers of lactic and glycolic acids,    -   polyanhydrides,    -   polydimethylsiloxane,    -   poly(ε-caprolactone) and its derivatives,        poly(β-hydroxybutyrate, poly(hydroxyvalerate) and        (β-hydroxybutyrate-hydroxyvalerate) copolymers, or poly(malic        acid),    -   amphiphilic block polymers of poly(lactic acid)-poly(ethylene        oxide) type,    -   saturated polyesters (poly(ethylene terephtalate)),    -   polyanhydrides, polyorthoesters and polyphosphazenes.

These synthetic polymers, biopolymers or derivatives thereof which arechosen in order to be effective matrix forming and/or coating agents,may exhibit a molar mass of greater than 10³ g/mol, preferably ofgreater than 2×10³ g/mol and more particularly of between 2×10³ and2×10⁶ g/mol. Preferably, the concentration of the polymer is under thesaturation concentration of the solvent by the polymer.

Solvent of the Discontinuous Phase

A suitable solvent according to the invention is a solvent which, undercertain conditions of pressure and temperature, is chemically inert withregard to the polymer, the divalent cationic ion or the activesubstance, and has physical and chemical properties that make itpossible to obtain an emulsion of solvent in CO₂ liquid and/orsupercritical.

The solvent is preferably a polar solvent which is able to solubilizethe substances, including the active substances, present in thediscontinuous phase.

It may be selected among protic or aprotic solvents, organic solvents orwater.

The solvent is preferably biocompatible.

As used herein “biocompatible” refers to those solvents which are,within the scope of sound medical judgement, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio.

Examples of suitable biocompatible solvents are notably water,tetraglycol, low molecular weight polyethylene glycol, propylene glycol,water and tetraglycol being particularly preferred.

Tetraglycol is also called glycofurol orTetrahydrofurfurylpolyethyleneglycol, Tetrahydrofurfuryl AlcoholPolyethyleneglycol Ether (CAS: 9004-76-6).

The weight of solvent may represent 0.02% to 20% of the weight ofsupercritical or liquid fluid.

Active Substance

The obtained particles can possess a biological activity, either becauseof the nature of the polymer from which they are formed or because theyadditionally incorporate an active substance.

In a preferred embodiment, the discontinuous phase further comprises oneor more active substance(s).

The active substance may be soluble or insoluble in the solvent of thediscontinuous phase. Preferably, the active substance is added in thesolution of the discontinuous phase before bringing it into contact withthe CO₂ phase.

As examples of active substances which may be contained in themicroparticles of the invention, mention may particularly be made,without limitation of pharmaceutical, cosmetic, diagnostic, veterinary,food-processing, phytosanitary active substance.

Other examples of active substances suitable according to the inventionare notably additives for plastics, synthetic rubbers, thermosettingresins, latex and dispersion, ink and textiles.

Examples of pharmaceutical products include notably, antipyretics,aspirin and derivatives, antibiotics, anti-inflammatories,antiulceratives, antihypertensives, neuroleptics, anti-depressants,analgesics, antifungics, antiviral, antitumorous agents,immunomodulators, antiparkinsonian, nucleotides, oligonucleotides,peptides, proteins, radionucleides.

Examples of cosmetic active substances include notably self-tanning oranti-UV agents.

Examples of processed foodstuffs are notably vitamins.

Examples of veterinary products include notably hormones, vaccines,anti-inflammatories, antibiotics.

Examples of phytosanitary active substances are notably herbicides,bactericides, fungicides, insecticides, acaricides or regulators ofgrowth.

It is also possible to incorporate, in the particles, compounds with adiagnostic purpose. They can thus be substances emitting electromagneticrays or substances detectable by X-ray, ultrasound or nuclear magneticresonance. The particles can thus include iron oxyde particles, such asmagnetite or maghemite, gadolinium chelates, radio-opaque materials,such as, for example, air or barium, or fluorescent compounds, such asrhodamine or nile red, or gamma emitters, for example indium ortechnetium.

Surfactants

The emulsion droplets according to the invention can be stabilized byusing surfactant molecules. Surfactants may indeed reduce theinterfacial tension (γ) and consequently the interfacial free energy(GS).

Preferably, the surfactant has a cloud point pressure and a cloud pointtemperature which are respectively lower to the conditions of pressureand superior to the conditions of temperature used in the methodaccording to the invention.

The surfactant may be solubilized in the CO₂ phase before bringing theliquid (i.e. the solvent containing a substance selected from the groupconsisting of a polymer, a divalent cationic ion or a mixture thereof)intended to form the discontinuous phase into contact with it.

As an example of suitable surfactants, mention may be made of the grouphaving, fluorocarbones, polydimethylsiloxanes, siloxanes andpolycarbonates tails.

Preferably, the fluorocabone surfactant is a compound of formula (I)

whereinR_(f) is a poly- or perfluoro-(C₄-C₁₂)alkyl,n is 1 to 15.

Such surfactants have been disclosed in Riess et al. (U.S. Pat. No.5,344,930) and Krafft et al., 1990.

Advantageously, the aqueous droplets once formed in the CO₂ phase arethen protected against coalescence due to the great elasticity of theinterfacial layer formed by the surfactant (Tewes et al., 2004).

Preferably, the surfactant is a compound of formula (I) wherein R_(f) isa C₈-perfluoroalkyl group and n is 5 (called 5-(F-octyl)pentyl][2′-N,N,Ntrimethylaminoethyl]phosphate) or F₈C₅PC, or 11 (called11-(F-octyl)-undecyl][2′-N,N,N trimethylaminoethyl]phosphate) orF₈C₁₁PC.

Formation of Particles Via a Water-in-Carbon Dioxide Emulsion

According to a particularly preferred embodiment the solvent of thediscontinuous phase is water.

The water preferably further comprises an aqueous buffer, which meanssalts having a buffering power.

Examples of suitable buffer include notably TRIS (Tris(Hydroxymethyl)Aminomethan) buffer, phosphate or bicarbonate buffer, glycin/NaClbuffer. The concentration of these buffers is such that the pH in theaqueous droplets of the emulsion can be maintained in a range of 7 to10, during the solidification process. Typically, the concentration mayrange from 0.1 mol/L to 1 mol/L.

In a preferred aspect of the invention, the polymer is a biopolymer orderivative thereof, notably a protein or a polysaccharide, for examplehyaluronic acid or ovalbumin.

In one embodiment, the solidification of the discontinuous phase in stepii) comprises the addition of a reticulating agent.

The reticulating agent is preferably soluble in the liquid orsupercritical CO₂.

Examples of suitable reticulating agents are notably glyceraldehyde,formaldhehyde, glutaraldehyde, terephtaloyl chloride, epichloridrine,genipin, enzyme like transglutaminase, tyrosinase, diamine oxydase,peroxydase.

The solidification process seems to result from an interfacial reactionbetween the reticulating agent, which is present in the continuousphase, and the biopolymer which is present in the discontinuous phase.

According to another preferred embodiment, the discontinuous phasefurther comprises a divalent cationic ion M²⁺.

As examples of divalent cations, mention may be made of Ca²⁺, Fe²⁺,Mg²⁺, Mn²⁺, Zn²⁺, Li²⁺, Al²⁺, Ni²⁺, Cd²⁺, Ti²⁺, Co²⁺, Ba²⁺, Cs²⁺, Cu²⁺;among these Ca²⁺ is particularly preferred.

As a source of divalent cationic ion, metal hydroxides may be used. Asan example, calcium hydroxide (CaOH₂) may be used as a source of Ca²⁺.

The concentration of the divalent cationic ions is preferably close tothe saturation concentration of M²⁺ in the solvent at the pH of thediscontinuous phase, at least at the beginning of the solidificationprocess. Typically, the concentration of M²⁺ may vary from 0.5 to 5% byweight of the total weight of the discontinuous phase.

Preferably, the pH in the droplets of the discontinuous phase allows theformation of CO₃ ²⁻ ions from the solvatation of CO₂ molecules in thediscontinuous phase. Preferably, the pH ranges from 7 to 10 and morepreferably from 8 to 9.

According to a possible mechanism, the inventors have shown that theMCO₃ particles production results from two competitive phenomena: theemulsification of the aqueous solution by the CO₂ phase and theformation of MCO₃ solid crystals.

The MCO₃ crystallization phenomenon is performed in several steps.

Firstly, the diffusion of the CO₂ molecules into the alkali bufferedsolutions is accompanied by the formation of several ionic species dueto its combination with the HO⁻ ions and, if the pH decreases, with theH₂O molecules, as defined by the following equations:

The proportion of the different species present in the solution dependson the pH. In the alkaline region, CO₃ ²⁻ and HCO₃ ⁻ speciespredominate.

Secondly, the CO₃ ²⁻ ions react with the M²⁺ ions, leading to theformation of MCO₃.

For example, CaCO₃ salts which have a very low aqueous solubility(˜0.0013 g in 100 g of water) crystallize under various forms (calcite,aragonite, vaterite, hydrates . . . ) depending on the initialconditions (Kitamura et al.; 2002; Hostomsky, 1991; Tai, 1993; Tai,1995; Kitamura, 2002).

The pH may also influence the formation of MCO₃ particles.

Indeed, CO₃ ²⁻ ions, which contribute to the precipitation process, areonly present from a pH higher than 8 (Rigopoulos et al., 2003).

This implies that, after the occurrence of a pH diminution in theprocess, induced by the combination of the CO₂ with HO⁻ or H₂O([Eq]1-3), the CO₃ ²⁻molecules are no longer present, and the MCO₃precipitation is terminated. Moreover, the acidic pH leads to the MCO₃dissolution.

On another hand, the pH of the solution also controls the M²⁺concentration due to the reaction between the M²⁺ and the HO⁻, leadingto the formation of solid M(OH)₂ when the ionic product (IP) is higherthan the solubility product constant (Ks) of the reaction, as describedby the equation 5. Therefore, the formation of M(OH)₂ is enhanced whenthe pH increases, leading to a decrease in the M²⁺ concentration.

Consequently, a too high pH decreases the M²⁺ concentration in theaqueous solution, whereas a too low pH leads to the decrease in the CO₃²⁻ concentration. These two species are the basis of the crystallizationprocess and their presence strongly depends on the pH. Therefore, it isadvantageous to precisely and strongly buffer the aqueous solution atthe right pH value, in order to control the process.

The emulsification process results in the formation of aqueous dropletsdispersed in the CO₂ phase.

In the formation of MCO₃ particles, the inventors have demonstrated thatthe pressure of CO₂ both play a role of discontinuous phase and ofreactant leading to the solidification of the discontinuous phase.

Indeed, it is particularly preferred that the emulsification step occursbefore the crystallization, as emulsion droplets are the templates thatcontrol the crystallization and thus, the particle formation. When asurfactant is present, more quickly the surfactant molecules areadsorbed at the interface, more quickly stable droplets are formed. Thisaspect is favoured by the low viscosity of the CO₂ phase in theconditions according to the invention, which enhances the adsorptionrate of the surfactant molecules.

The crystallization and emulsification phenomena are both controlled bythe CO₂ pressure in an opposite way. In fact, the increase in the CO₂pressure leads to an increase in the emulsification efficiency andemulsion stability due to the diminution in the difference between thedensities of the CO₂ and aqueous phases and due to the reduction of theinterfacial tension.

On the other hand, the increase in the CO₂ pressure leads to an increasein the solubility of the CO₂ in the aqueous phase, due to theequilibration of the CO₂ fugacity between the gas CO₂ phase and thewater phase. Unfortunately, an excess of CO₂ dissolved in the aqueousphase results in MCO₃ decomposition due to the decrease of the pH asdescribed in equation 6.

Therefore, the CO₂ pressure is preferably precisely controlled in arange of pressures allowing the obtaining of the most stable W/Cemulsion, controlling in the same time the quantity of dissolved CO₂ inthe water phase.

The CO₂ pressure can be adapted according to the concentration of thebuffer molecules in the discontinuous phase in order to maintain a pHallowing the latest reaction.

The particles of polymer MCO₃ prepared according to the invention aregenerally microspheres.

The morphology of particles of polymer/MCO₃ may vary according to the pHof the aqueous droplets of the emulsion and to the nature of the polymerand cation used.

The difference in the particles shape obtained at pH 9 to 8 can resultfrom a competitive effect between the crystallization and emulsificationphenomena.

The polymer being generally homogeneously solubilized in the aqueousdroplet, the crystallization of MCO₃ is generally uniform, leading tospherical microspheres.

The particles obtained from a water in carbon dioxide emulsion can belyophilized, notably after their washing.

Typically, the particles formed via a water-in-CO₂ emulsion have anarrow size distribution, with a mean diameter by volume or by numberranging from 0.1 μm to 10 μm.

Formation of Particles Via an Organic Solvent in Carbon Dioxide Emulsion

In a further aspect of the invention, the discontinuous phase is anorganic solvent.

Suitable organic solvents according to the invention are solvents whichbecome, for given conditions of pressure and temperature and/or inadmixture with another solvent, miscible with either liquid orsupercritical CO₂, thus allowing its extraction and the solidificationof the polymer.

The organic solvent is preferably also partly or highly miscible withwater.

Examples of suitable solvents are notably, tetraglycol, low molecularweight polyethylene glycol, propylene glycol.

Preferably, the solvent is tetraglycol.

Preferably, the polymer is a synthetic polymer, more preferably apolymer of lactic acid or a copolymer of lactic acid and glycolic acid.

According to this aspect of the invention, the solidification of thediscontinuous phase is performed by extracting the solvent from thediscontinuous phase, thus desolvating the polymer and solidifying thediscontinuous phase.

In one embodiment of the invention, the solvent is extracted by adding asecond solvent, said second solvent being miscible with the solvent ofthe discontinuous phase and not being a solvent of the substancescontained in the discontinuous phase, thereby forming a mixture ofsolvents which is miscible in the continuous phase.

Preferably, the second solvent is water or propylene glycol

In another embodiment, the solvent is extracted from the discontinuousphase by bringing the supercritical CO₂ in liquid state. This can bedone by reducing the temperature and the pressure so as to desolvate thepolymer in a controlled way. When temperature is lower than Tc, thepressure may be increased to enhance the organic solvent extraction.

The expression “in a controlled way” is understood to mean the fact thatthe system is always under conditions close to equilibrium and is notsubjected to sudden variation in pressure.

Preferably, the solvent is extracted by contacting a flow of freshliquid CO₂.

Preferably, the temperature of liquid CO₂ ranges from 10° to 20° C. andis more preferably of about 15° C.-16° C.

Preferably, the pressure of liquid CO₂ ranges from 80 to 160.10⁵ Pa andmore preferably from 100 to 150.10⁵ Pa.

According to a further embodiment, the solvent is extracted by bothadding said second solvent and bringing the supercritical CO₂ in liquidstate.

After a substantial amount of solvent is extracted, an aqueous solutionof a dispersing agent may advantageously be added so as to harden theobtained particles.

Examples of suitable dispersing agents are notably triblock copolymersof polyoxyethylene-polyoxypropylene-polyoxyethylene known as poloxamersor Pluronic®.

Preferably, the dispersing agent is the poloxamer sold under thetradename of Pluronic F68, which is agreed by FDA.

Preferably, the method according to the invention is carried out in aclosed reactor, such as an autoclave.

The obtained particles may be recovered by using conventional methodssuch as filtration or centrifugation and can be lyophilized after theirwashing.

In a further aspect, the invention is directed to dispersions ofparticles obtainable by the method of the invention. Preferably, thedispersion results from a water-in-carbon dioxide emulsion.

In a still further aspect, the invention is directed to particlesobtainable by the method of the invention.

Preferably, the particles are microspheres or nanospheres comprising abiopolymer, MCO₃ and an active substance dispersed in a MCO₃/biopolymermatrix system. The particles may notably comprise an active substancecoated with a biopolymer or a biodegradable synthetic polymer.

Typically, the mean diameter by volume or by number of the obtainedparticles ranges from 0.1 μm to 100 μm, preferably from 1 μm to 20 μmand more preferably from 2 μm to 5 μm.

In a still further aspect, the invention is directed to the use ofparticles or dispersions thereof as a vehicle for cosmetic,pharmaceutical, diagnostic, veterinary, phytosanitary or foodstuffproducts.

More particularly, the particles according to the invention are usefulto encapsulate active substances and notably to prepare controlled drugdelivery systems, such as fast or slow drug-release formulations.

As a result of the immunogenicity of certain biopolymers such asovalbumin, the particles of the invention may be specifically capturedby the cells of the immune system (macrophages) and thus may beparticularly useful in the field of antitumoral vaccination or ofcellular imaging.

As an example, the particles able to be captured by the immune systemcells may contain proteins coming from tumours or a contrast agent as anactive substance.

The particles or the dispersions thereof may also be used in furtherdiverse applications such as:

-   -   in paper as filler or as a retaining agent,    -   in adhesives,    -   in inks,    -   as anticorrosion coatings,    -   fragrance compositions in textiles or cosmetics.

FIGURE

FIG. 1: Schematic representation of the equipment used to formulateCaCO₃ particles in CO₂ medium. A: Bottle of CO₂; B: cooling system forthe CO₂; C: pump for the CO₂; D: heating system; E: autoclave; F: HPLCpump.

EXAMPLES 1. Materials

Calcium hydroxide; chicken egg white albumin of grade V (OVA); bovineserum albumin-fluorescein isothiocyanate (BSA-FITC) were purchased fromSigma Aldrich, France. Tris(hydroxymethyl)aminomethan (TRIS) waspurchased from Merck, France. The CO₂ used in the experiments is ofmedical grade (purity 99.99%) and was purchased from Air Liquide,France. Ultrapure water was produced by a MilliQ plus 188 apparatus(Millipore, France).

The phosphocholine-based fluorinated surfactants were synthesizedaccording to Krafft et al., 1990. Their purity (>99%) was assessed byHPLC, NMR and elemental analysis.

The BSA (Bovine Serum Albumin) was purchased from Sigma (France).

Pluronic F68®, Lutrol F68® (poloxamer 188)(HO—(C₂H₄O)₈₀—(C₃H₆O)₂₇—(C₂H₄O)₈₀—H) were provided by BASF

Poly(α-hydroxyacid)s such as PLA50 and PLGA 37.5/25 were purchased fromPhusis (France)

Glycofurol was provided by Sigma (France)

Propylene glycol was provided by Sigma (France).

2. Methods

2.1. Microsphere Preparation

The experimental work was conducted using the apparatus presented inFIG. 1 (Separex Equipments, Champigneulles, France).

It is composed of a 500 mL autoclave having at its upper side a stirrercoupled to an internal Teflon blade. This stirrer allows mixing thecontent of the cell with a maximal rate of 1500 rpm. A known quantity ofCO₂ was introduced from the top of the cell at the desired pressure andtemperature. The polymer solution was brought from the bottom of thecell under a controlled flow rate and at a pressure higher than the onealready existing in the autoclave.

2.2. Microsphere Size and Morphology

2.2.1. Particles Prepared from a Water-In-CO₂ Emulsion

The different kinds of microparticles were observed in aqueoussuspension by an optical microscope (Olympus type BH-2) coupled to a CCDcamera (COHU). Image captures and particles size determination wereperformed with the Archimed 3.9.0 software (Microvision instruments,Evry, France).

Scanning electron microscope (SEM; JEOL 6301F; Jeol France, 78000Croissy-sur-Seine), equipped with an energy dispersive X-ray (EDX) unit,was utilized to examine the surface morphology, as well as for having anidea of the size distribution of the microspheres. Lyophilized samplesof microspheres were mounted on aluminum stubs and sputter coated with acarbon layer. A voltage of 5 or 20 kV was applied.

Size measurements were performed using a Coulter® counter.

2.2.2. Particles Prepared from an Organic Solvent in CO₂ Emulsion

The different kinds of microparticles were observed in aqueoussuspension by an optical microscope (Olympus type BH-2) coupled to a CCDcamera (COHU). Image captures and particles size determination wereperformed with the Archimed 3.9.0 software (Microvision instruments,Evry, France).

Scanning electron microscope (SEM; JEOL 6301F; Jeol France, 78000Croissy-sur-Seine), equipped with an energy dispersive X-ray (EDX) unit,was utilized to examine the surface morphology, as well as for having anidea of the size distribution of the microspheres. Lyophilized samplesof microspheres were mounted on aluminum stubs and sputter coated with acarbon layer. A voltage of 5 or 20 kV was applied.

2.3. Composition of Spheres Prepared from a Water-In-CO₂ Emulsion

The elementary composition of the microspheres was determined byrecording the energy dispersive X-ray (EDX) spectra collected on 60seconds at different locations. The measurements were performed with thesame apparatus as the one used for SEM experiments. The detection limitof the measurements is of 1 wt %.

The composition of the washed and lyophilized microspheres wasinvestigated by FT-IR spectroscopy. The spectra were recorded between4000-500 cm⁻¹ with a Bruker FT-IR Vector 22 using potassium bromidepellets. For each spectrum, a 20-scan interferogram was collected at aresolution of 2 cm⁻¹. A pre-recorded water vapour absorption spectrumunder identical conditions was subtracted after data collection. Priorto obtaining the sample spectrum, an open beam background spectrum wasrecorded.

2.4. Particle Zeta Potential Measurements

The zeta potential (ζ) of various kinds of CaCO₃ particles was measuredunder a voltage of 100 mV using a Zetasizer 2000 (Malvern). This valuewas measured for suspensions having approximately the same particleconcentration. A single point measurement was conducted for a pH of 8and under a conductivity of 400 μS/cm, adjusted by a 0.5 M TRIS buffer.Each value represents the average between fifteen measurements.

2.5. FITC-BSA Microsphere Loading

In order to know if it is possible to encapsulate proteins into theparticles, and in same time, to visualize the protein localization inthe particles, the particles were formulated in presence of afluorescent protein (BSA-FITC). The technique was identical to the onedescribed in section 2.1. and in examples 1 to 3 hereafter, with thesingle difference that in the initial alkali aqueous solution containing1 g/L of OVA, 5 mg/L of BSA-FITC were added. The particles were analyzedby fluorescent microscopy (Zeiss Axiotop 2 MOT, UV Zeiss type Atto Arc 2HBO 100W lamp) with an excitation wavelength of 488 nm and an emissionwavelength of 519 nm, corresponding to the FITC-labelled substrate.

2.6. Biocompatibility Study

The complement system is involved in the opsonisation of the foreignobject. The activation of the complement system by the microparticleswas studied by a technical method called “CH50 modified method” (Mayeret al., 1961; Kazatchkine et al., 1986; Passirani et al., 1998).

3. Examples 1 to 3 Particles of Biopolymers 3.1. Examples 1 and 2Particles of CaCO₃/Biopolymers

Step 1: Solubilization of the Surfactant in the CO₂ Phase (OptionalStep)

The surfactants which were used in this procedure for the formation ofparticles of CaCO₃ were fluorinated surfactants such as F₈C₅PC orF₈C₁₁PC (Prof. M P Krafft, Institut Charles Sadron, Strasbourg, France)(Riess et al., U.S. Pat. No. 5,344,930; M. P. Krafft et al., 1990).

These surfactants have a fluorinated chain, a phosphocholin group and alinker comprising 5 or 11 carbon atoms.

An amount of surfactant (0 to 50 mg) was added in the reactor of 500 ml,thermostatically controlled at 25° C.

Under stirring with a propeller or anchor moving blade (300-400 rpm),the CO₂ was introduced in the autoclave up to a pressure of 200.10⁵ Pa.This step of solubilization of surfactant was performed during at least10 minutes.

Remark: This step is optional since it is possible to formulateparticles of CaCO₃ and of biopolymer without surfactant.

Step 2: Addition of the Aqueous Phase

The aqueous phase was composed of 3 elements:

a) an aqueous buffer to maintain the pH in a determined range (basicpH=8-10),

2 types of buffer were used to maintain the basic pH at concentrationranging from 0.5 to 1 mol/L: TRIS buffer, and Glycin/NaCl buffer;

b) calcium hydroxide (CaOH₂) as a source of Ca²⁺ for the formulation ofcalcium carbonate in concentrations which were comprised between 0.5 and1%;

c) a biopolymer which plays a role of matrix wherein the crystallizationof CaCO₃ occurs and which allows to structurate the final form of themicroparticle.

Ovalbumine grade VII (protein) and hyaluronic acid (polysaccharide) atconcentrations of 1 g/L were used in these experiments. The addition ofthe aqueous phase was effected in one or two steps.

-   -   Addition in one step: once the surfactant was completely        solubilized, 25 mL of aqueous solution containing the three        elements (a), (b) and (c) were added in the autoclave under        continuous stirring at 1000 rpm at a flow rate of 10 mL/min        leading to a final pressure in the cell ranging form 220 to        240.10⁵ Pa. After adding the total volume, the stirring was then        continued during 5 minutes because for larger times, the pH        decreases to a level that results in the decomposition of the        CaCO₃ particles into a soluble salt.    -   Two steps addition: once the surfactant was completely        solubilized, 25 mL of a solution of a biopolymer at 1 g/L in a        buffer solution were added in the autoclave with a flow rate of        10 mL/min and by setting the stirring rate at 1000 rpm. The        stirring was continued during 5 minutes then 15 mL of a solution        of Ca(OH)₂ at 1% in an identical buffer were then administered        in the reactor. The speed rate was continued at a stirring rate        of 1000 rpm during 5 minutes. The final pressure in the        autoclave was comprised between 240 and 260 bars.

Step 3: Depressurizing the Autoclave

At the end of the processes, the stirring was stopped and the autoclavewas slowly depressurized (−40 to 50.10⁵ Pa/min).

Step 4: Recovering the Microparticles

The suspension of microparticles was centrifuged at 4000 rpm during 15minutes.

The centrifugation pellet was taken with 40 mL of demineralised water,then centrifuged again at 4000 rpm during 15 minutes. This rinse waseffected 3 times. The centrifugation pellet of microparticles wasfinally taken with 2 mL of demineralized water and lyophilized to obtaina dry powder of microparticles.

Example 1 Particles of CaCO₃/Ovalbumine Grade (VII)

The above procedure was applied with the following conditions.

-   -   Formulation        -   10 mg of surfactant F₈C₅PC        -   25 mL of aqueous solution comprising Ca(OH)₂ at 0.5% and            Ovalbumine grade VII 1 g/L in a TRIS buffer 0.5 M, pH=8.4.    -   Process        -   Addition in one step        -   Final pressure=235.10⁵ Pa    -   Yield        -   Mean yield=115 mg per formulation (by autoclave)    -   Measurement of the size with Coulter® Multisizer

This method used to determine the particles diameter is based on thevariation of impedance when a particle goes through between 2electrodes.

The measurement of the size was effected after dispersing the particlesin a ISOTON-Tween 1% medium.

The statistical results were as follows

TABLE 1 Statistical results of the measurements of the size withCoulter ® Multisizer for the studied formulation. Mean (μm) 2.92Standard deviation (μm) 2.67 d 10 (μm) 0.87 d 50 (μm) 1.97 d 90 (μm)6.38

-   -   Differential Scanning Calorimetry (DSC)

A DSC analysis was realized on the formulation and on each of thecomponents of the microparticle, that is ovalbumin, CaCO₃, and thefluorinated surfactant. The DSC profile of the microparticles displayeda signal around 155-158° C., a signal that corresponds to ovalbumin VII.The signal corresponding to the surfactant was not detected as it waspresent in too low amounts.

This study thus allowed to conclude that the formulated particlescontain ovalbumin in their structure.

-   -   Study of the activation of the complement (biocompatibility        study).

Results obtained by measuring the CH50 consumption versus surface showedthat CaCO₃ particles were significantly less active than controlparticles (sephadex G50).

Example 2 Particles of CaCO₃/Hyaluronic Acid

Particles of CaCO₃/Hyaluronic Acid were prepared according to example 1,with or without the surfactant F₈C₁₁PC.

The results obtained with the Coulter® Multisizer showed that the use ofa surfactant had no influence in the size of the particles. Theparticles obtained without surfactant display a mean diameter 2 timessuperior than those obtained with a surfactant.

TABLE 2 Statistical results of the measurements of the size withCoulter ® Multisizer for formulations of microparticles CaCO₃/Hyaluronicacid with or without surfactant. Formulation with a Formulation withoutsurfactant surfactant Mean (μm) 2.40 5.86 Standard deviation (μm) 2.254.05 d 10 (μm) 0.85 1.52 d 50 (μm) 1.05 4.70 d 90 (μm) 5.99 12.2

It was experimented to encapsulate BSA-FITC into CaCO₃ particles inorder to demonstrate the location of the protein inside or at thesurface of the microspheres, by using the method described in section2.5. with OVA as template for particle obtaining and by buffering the pHat a value of 8. According to the analysis of the transmission opticalmicrograph, the obtained particles had a size of about 1 μm and kept thedistribution obtained without BSA-FITC. Further, according to thefluorescence optical micrograph, the protein was encapsulated in themicrospheres as revealed by the fluorescent spots, which coincide withthe microspheres of the transmission optical micrograph. Identicallyencapsulation was obtained in the absence of two surfactants or byproceeding with a one step or two steps addition of the aqueoussolution.

3.2. Example 3 Microparticles of Ovalbumin

3.2.1. Microparticles Preparation

Microparticles of ovalbumin were prepared according to the followingmethod.

Step 1: Solubilization of the Surfactant in the CO₂ Phase

10 to 20 mg of fluorinated surfactant was added in the autoclave of thereactor, thermostatically controlled at a temperature of 40° C.

The CO₂ was then introduced in the autoclave under stirring with amoving blade up to a pressure of 270.10⁵ Pa.

Step 2.: Addition of the Aqueous Phase

Once the surfactant was completely solubilized, 1 mL of a ovalbuminsolution was added under continuous stirring (1500 rpm, at a flow rateof 10 mL/min leading to a final pressure ranging from 200 to 300.105Pa). After adding the total volume, the stirring was continued during 1hour at 1500 rpm.

Step 3.: Addition of Reticulating Agent

1 mL of glyceraldehyde under a flow rate of 10 mL/min or 20 mg ofterephtaloyl chloride was added in the autoclave in order to reticulatethe formed particles. In the case of terephtaloyl chloride, theovalbumin solution in step 2 was buffered around a pH of 10.

Step 4.: Depressurizing the Autoclave

At the end of the process, the stirring was stopped and the autoclavewas depressurized slowly (30-50.10⁵ Pa/min).

Step 5.: Recovering the Microparticles

The suspension of microparticles was centrifuged at 4000 rpm during 15minutes.

The centrifugation pellet was taken with 40 mL of demineralized water,then centrifuged again at 4000 rpm during 15 minutes. This rinse waseffected three times. The centrifugation pellet of microparticles wasfinally taken with 2 mL of demineralized water and lyophilized to obtaina dry powder of microparticles.

3.2.2. BSA-FITC Encapsulation

The method of encapsulation disclosed in part 2.5 was applied with thefollowing conditions:

-   -   Ovalbumin solution: 4 g/L+5 mg/L BSA-FITC in PBS pH=7.4    -   10 to 20 mg of fluorinated surfactant    -   Initial pressure 210.10⁵ Pa    -   Temperature=40° C.    -   Stirring rate=1500 rpm    -   1.5 mL of a solution ovalbumin and BSA-FITC    -   Final pressure of 280.10⁵ Pa    -   Time stirring=1 hour.    -   Reticulating agent (glyceraldehyde or terephtaloyl chloride)

By analyzing the transmission optical micrograph in visible light and influorescence (520 mm), fluorescent spots were observed in the particles,thus demonstrating the encapsulation of BSA-FITC.

3.2.3. Gadoteric Acid Encapsulation

The method of encapsulation disclosed in part 2.5 was applied with thefollowing conditions:

-   -   Ovalbumin solution: 4 g/L    -   10 to 20 mg of fluorinated surfactant    -   Initial pressure 210.105 Pa    -   Temperature=40° C.    -   Stirring rate=1500 rpm    -   0.8 mL of ovalbumin 0.4 g/L mixed with gadoteric acid at 0.1        mol/L    -   Final pressure of 280.105 Pa    -   Time stirring=1 hour.    -   Reticulating agent: 0.4 mL of glyceraldehyde

The obtained particles were then analyzed with an optical microscope.The particles observed were spherical with a size ranging from 1 to 10μm.

From scanning electron microscopy spherical particles exhibited a pleatsurface. From EDX analysis, gadolinium was mainly encapsulated into theparticles.

4. Examples 4 and 5 Microparticles of Biodegradable Synthetic Polymers4.1. Example 4 Microparticles of Biodegradable Polymers Prepared from anEmulsion of Tetraglycol in Supercritical CO₂: Solidifying theDiscontinuous Phase Droplets by Adding Water

4.1.1. Microparticles Preparation

Step 1.: Solubilization of Polymer in Tetraglycol

The polymer was a polymer of lactic acid (PLA) or a copolymer of lacticand glycolic acid (PLGA) or any polymer soluble in the tetraglycol.

The polymer was solubilized in tetraglycol in order to obtain a solutionof low viscosity. The percentage of polymer in the solution will dependon the molecular weight of polymer and of its solubility in tetraglycol.

Typically, a PLGA of a mean, molecular weight of 20000 g/mol wassolubilized in a ratio of 2.5% to 20% (w/v) (weight/volume) intetraglycol.

Step 2.: Emulsion=Solution of Polymer/Tetraglycol Dispersed in CO₂

20 mL of the solution of polymer in tetraglycol were introduced in areactor thermostatically controlled of 500 mL at 35° C.

Under stirring with a moving blade, CO₂ was added in the autoclave up toa pressure of 180.10⁵ Pa. These experimental conditions allowed toobtain an emulsion of the solution of polymer in supercritical CO₂.

This step, which was necessary to form and to stabilize the droplets ofthe solution of polymer in tetraglycol, was performed during 5 minutes.

The time of this step depended on the percentage of the polymersolubilized in tetraglycol.

Step 3. Extraction of the Organic Solvent

50 mL of the aqueous solution at 1% (w/v) of Pluronic F68 was then addedat a flow rate of 10 mL/min in the autoclave. The pressure in theautoclave was then of 280.105 Pa. The addition in the solution of thedispersity agent was:

-   -   to provide the extraction of tetraglycol, by favouring the        precipitation and the hardening of the polymer;    -   to prevent the agglomeration of microparticles by the presence        of the dispersing agent Pluronic F68.

Step 5.: Depressurization of the Autoclave

The autoclave was then slowly depressurized (about 30-50.10⁵ Pa/min).

25 mL of an aqueous solution at 1% (w/v) of Pluronic F68 was finallyadded at a flow rate of 10 mL/min in the autoclave once at theatmospheric pressure.

Step 6.: Recovering Microparticles

The suspension of microparticles was centrifuged at 4000 rpm during 20minutes.

Each pellet of microparticles was taken in 45 mL of demineralised water,then centrifuged again at 4000 rpm during 10 minutes. This rinse wasperformed two or three times.

The pellet of microparticles was finally taken in 2 mL of demineralisedwater and lyophilized to obtain a dry powder of microparticles.

4.1.2. Microparticles Characterization

Microparticles obtained from a solution of PLA 50 10% (w/v) in thetetraglycol or from a solution of PLGA 37.5/25 5% (w/v) in tetraglycolwere observed by an optical microscope coupled with a software(Microvision® allowing the size estimation. These microparticles had aestimated diameter comprised between 1 μm to 10 μm.

4.2. Example 5 Microparticles of Biodegradable Synthetic Polymers froman Emulsion of Tetraglycol in Supercritical CO₂ Solidifying theDiscontinuous Phase Droplets by Extraction with Liquid CO₂ and then byAddition of Water

4.2.1. Microparticles Preparation

Step 1.: Solubilization of Polymer in Tetraglycol

The polymer was a polymer of lactic acid (PLA) or a copolymer of lacticand glycolic acid (PLGA) or any polymer soluble in the tetraglycol.

The polymer was solubilized in tetraglycol in order to obtain a solutionof low viscosity. The percentage of polymer in the solution will dependon the molecular weight of polymer and of its solubility in tetraglycol.

Typically, a PLGA of a mean, molecular weight of 20000 g/mol wassolubilized in a ratio of 2.5% to 20% (w/v) (weight/volume) in thetetraglycol.

Step 2.: Incorporation of the Protein in the Solution

The BSA (Bovine Serum Albumine was chosen as model. It was spheronizedin the presence of Lutrol F68® according to the technique disclosed byMorita et al., 2000.

The co-lyophilizate BSA-Lutrol F68 was added to the solution ofpolymer-tetraglycol. The Lutrol F68 can be solubilized at 37° C. intetraglycol allowing to obtain a suspension of particles of BSA in thesolution of PLGA/Lutrol F68/tetraglycol.

Step 3.: Emulsion of a Solution of Polymer/Tetraglycol in SupercriticalCO₂

10 mL of the suspension of BSA in the solution of polymer/LutrolF68/tetraglycol were introduced in the reactor of 500 mL thermoregulatedat 35° C. Under stirring with a propeller or anchor moving blade, theCO₂ is introduced in the autoclave up to a pressure superior or equal to240.10⁵ Pa. and inferior or equal to 300.10⁵ Pa. These experimentalconditions allow to obtain an emulsion of a solution ofpolymer/tetraglycol in the CO₂ in supercritical conditions.

This step, which allows the formation and the stabilization of dropletsof solution polymer/tetraglycol containing particles of BSA, wasperformed during 10 to 12 nm, time necessary to obtain a pressure of240.10⁵ Pa. in the autoclave.

Step 4.: Extraction in CO₂ Liquid Phase at 15° C.

While maintaining the stirring, the temperature of the middle wasdecreased at 15° C., the CO₂ then became liquid. The decrease of thetemperature of the middle led to a decrease of the pressure which wasstable between 100 and 150.10⁵ Pa. when the temperature of the middlewas at 15-16° C. Under these conditions of pressure and temperature, theCO₂ was in liquid state, allowing an extraction of the part oftetraglycol and the precipitation of the polymer as microparticles,around particles of BSA.

The extraction phase (at 15° C. and 100-150.10⁵ Pa.) was performedduring at least 30 nm.

Step 5.: Microparticles Curing

25 or 50 mL of an aqueous solution containing 1% (w/v) of Pluronic F68were then added to a flow of 10 mL/mn in the autoclave. The pressure inthe autoclave was then at least of 200.10⁵ Pa.

The addition of an aqueous solution of surfactant aimed at:

-   -   achieve the extraction of tetraglycol, favourizing the        precipitation and the hardening of the polymer,    -   preventing the agglomeration of microparticles by the presence        of the surfactant Pluronic F68.

Step 6.: Depressurizing the Autoclave

The autoclave was then slowly depressurized (about 30-50.105 Pa/min).

25 mL of an aqueous solution at 1% (w/v) of Pluronic F68 was finallyadded at a flow rate of 10 mL/min in the autoclave once at theatmospheric pressure.

The suspension of microparticles was then introduced in 100 or 150 mL ofdemineralised water under stirring to complete the extraction.

Step 7.: Recovering Microparticles

The suspension of microparticles was centrifuged at 4000 rpm during 20minutes.

Each pellet of microparticles was taken in 45 mL of demineralised water,then centrifuged again at 4000 rpm during 10 minutes. This rinse wasperformed two or three times.

The pellet of microparticles was finally taken in 2 mL of demineralisedwater and lyophilized to obtain a dry powder of microparticles.

4.2.2. Microparticles Characterization

Microparticles obtained from a solution of PLGA 37.5/25 5% (w/v) intetraglycol were observed by an optical microscope coupled with asoftware (Microvision®) allowing the size estimation. Thesemicroparticles had a estimated diameter comprised between 1 μm to 15 μm.

Under fluorescent microscopy FITC-BSA microparticles of PLGA 37.5/25showed a typical fluorescence indicating the incorporation of theprotein inside the particles.

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1. A method for preparing particles comprising the steps of: i)preparing an emulsion containing: as a continuous phase, supercriticalor liquid CO₂, and as a discontinuous phase, a solvent containing asubstance selected from the group consisting of a polymer, a cationicdivalent ion or a mixture thereof, said substance being soluble in saidsolvent and insoluble in said continuous phase, ii) solidifying saiddiscontinuous phase, thereby forming particles.
 2. The method of claim1, wherein, in step i) the emulsion contains as a continuous phasesupercritical CO₂.
 3. The method of claim 1, wherein the solvent isbiocompatible.
 4. The method of claim 1, wherein the discontinuous phasefurther comprises an active substance.
 5. The method of claim 4, whereinthe active substance is selected from pharmaceutical, diagnostic,cosmetic, veterinary, phytosanitary products, or processed foodstuffs.6. The method of claim 1, wherein the polymer is a biopolymer orderivatives thereof, or a synthetic polymer.
 7. The method of claim 6,wherein the biopolymer is or derives from a peptide, a protein, apolysaccharide or nucleic acids.
 8. The method of claim 6, wherein thesynthetic polymer is a biodegradable polymer.
 9. The method of claim 7,wherein the solvent is water.
 10. The method of claim 9, wherein thewater further comprises an aqueous buffer.
 11. The method of claim 9,wherein the solidification of the discontinuous phase comprises theaddition of a reticulating agent.
 12. The method of claim 9, wherein thediscontinuous phase further comprises a divalent cationic ion.
 13. Themethod of claim 12, wherein the divalent cationic ion is selected fromthe group consisting of Ca²⁺, Fe²⁺, Mg²⁺, Cu²⁺ Mn²⁺, Zn²⁺, Li²⁺, Al²⁺,Ni²⁺, Cd²⁺, Ti²⁺, Co²⁺, Ba²⁺, Cs²⁺.
 14. The method of claim 9, whereinthe emulsion further comprises a surfactant.
 15. The method of claim 8,wherein the solvent is an organic solvent.
 16. The method of claim 15,wherein the solvent is tetraglycol.
 17. The method of claim 15, whereinthe polymer is a polymer of lactic acid or a copolymer of lactic acidand glycolic acid.
 18. The method of claim 15, wherein thesolidification of the discontinuous phase is performed by extracting thesolvent.
 19. The method of claim 18, wherein the solvent is extracted byadding a second solvent, said second solvent being miscible with thesolvent of the discontinuous phase and not a solvent for the substancesof the discontinuous phase, thereby forming a mixture of solvents whichis miscible in the continuous phase.
 20. The method of claim 18, whereinthe solvent is extracted by bringing the supercritical CO₂ in liquidstate.
 21. The method of claim 15, wherein an aqueous solution of adispersing agent is added to the obtained particles.
 22. The method ofclaim 1, wherein the method is carried out in a closed reactor.
 23. Themethod of claim 1, wherein the obtained particles are recovered. 24.Dispersion of particles, obtainable by the method of claim
 1. 25.Particles obtainable by the method of claim
 23. 26. A vehicle ofcosmetic, pharmaceutical, diagnostic, veterinary or phytosanitary activesubstances or foodstuff products comprising a dispersion of particles ofclaim
 24. 27. Paper, inks, adhesives, anticorrosion coatings, fragrancecomposition, textiles comprising a dispersion of particles of claim 24.28. Particles comprising a biopolymer, MCO₃ and an active substance. 29.Particles of claim 28, wherein the mean diameter ranges from 1 μm to 20μm.
 30. A vehicle of cosmetic, pharmaceutical, diagnostic, veterinary orphytosanitary active substances or foodstuff products comprisingparticles of claim
 25. 31. Paper, inks, adhesives, anticorrosioncoatings, fragrance composition, textiles comprising particles of claim25.